New Species of Rhabdias (Nematoda: Rhabdiasidae) from Afrotropical Anurans, Including Molecular Evidence and Notes on Biology

Folia Parasitologica 57[1]: 47–61, 2010 © Institute of Parasitology, Biology Centre ASCR ISSN 0015-5683 (print), ISSN 1803-6465 (online) http://www.paru.cas.cz/folia/

New species of Rhabdias (Nematoda: Rhabdiasidae) from Afrotropical anurans, including molecular evidence and notes on biology

Kerstin Junker1, Nathaly Lhermitte-Vallarino2, Michela Barbuto3, Ivan Ineich4, Samuel Wanji5 and Odile Bain2

1 Department of Veterinary Tropical Diseases, University of Pretoria, Private Bag X04, Onderstepoort, 0110, South Africa; Current address: Onderstepoort Veterinary Institute, Private Bag X05, Onderstepoort, 0110, South Africa; 2 Muséum National d’Histoire Naturelle, UMR 7205 CNRS, Origine, Structure et Evolution de la Biodiversité, Parasitologie com- parée, CP52, 61 rue Buffon, 75231 Paris cedex 05, France; 3 Università degli Studi Milano Bicocca, Dipartimento di Biotecnologie e Bioscienze, Piazza della Scienza 2, 20126 Milano, Italy; 4 Muséum National d’Histoire Naturelle, UMR 7205, Origine, Structure et Evolution de la Biodiversité, Section Reptiles, Taxon- omie et Collections, CP 30, 25 rue Cuvier, 75005 Paris, France; 5 Research Foundation for Tropical Diseases and the Environment, P.O. Box 474, Buea, Cameroon

Abstract: Despite the small sample size the diversity of Rhabdias Stiles et Hassall, 1905 from anurans in the Afrotropical region was found to be high. Four species were collected from four localities, one in South Africa, two on Cameroonese mountains and one in Madagascar: Rhabdias picardiae sp. n. from the bufonid Amietophrynus gutturalis (Power); Rhabdias ohlerae sp. n. and Rhabdias tanyai sp. n. from the arthroleptids Leptopelis brevirostris (Werner) and Astylosternus rheophilus Amiet, respectively; and Rhabdias vencesi sp. n. from the mantellid Boophis madagascariensis (Peters). Distinctive characters between these species are numerous and obvious, based on body size, shape and size of the buccal capsule, arrangement of head papillae, and shape and size of the oesophagus and intestinal apex. Molecular data based on 500 bp of 12S rDNA and 600 bp of coxI of three of the four species are presented. Rhabdias vencesi resembles Rhabdias madagascariensis Chabaud, Brygoo et Petter, 1961 from an African ptychadenid introduced on Madagascar, but differs in body size and head morphology. The remaining new species are clearly distinct from those previously known from Afrotropical anurans. Outside the Afrotropics, some Rhabdias species present characters similar to those observed in the new species, but they all differ in various other characters. No clear correlation was seen between Rhabdias species and families of anuran hosts in this region. However, the narrow buccal capsule seen in Rhabdias species from Afrotropical lissamphibians opposes them to the majority of Rhabdias parasitic in chamaeleonids. Furthermore, the infective larva of R. vencesi has a conical pointed tail, while those of Rhabdias from chameleons have a rounded tail tip ornated with a few buds. Keywords: parasitic nematodes, Rhabdias, molecular characterisation, taxonomy, anurans, Amietophrynus, Astylosternus, Boophis, Leptopelis, Afrotropical region

Of the 39 species of Rhabdias Stiles et Hassall, 1905 Recently, renewed interest in the diversity of Rhabdias parasitising anuran hosts (Baker 1987a, Bursey et al. in the Afrotropical region led to detailed studies concen- 2003, Kuzmin et al. 2007, Martínez-Salazar and León- trating on hosts belonging to the Chamaeleonidae, a group Règagnon 2007, Martínez-Salazar 2008, Kuzmin and of saurian hosts found to harbour a wide variety of lung Tkach 2009), only three have been described in the Afro- worm species (Lhermitte-Vallarino and Bain 2004, Lher- tropical region, one from Ptychadenidae in Madagascar mitte-Vallarino et al. 2008, 2009a, b). (Chabaud et al. 1961), another from Arthroleptidae in During the past two years, lung worms were recov- Tanzania (Baker 1987b) and a third from two members of ered from four species of Afrotropical anurans belonging Bufonidae in South Africa (Kuzmin 2001). Close to this to the Bufonidae, Arthroleptidae and Mantellidae. Mor- region, in Egypt, Rhabdias was reported from a bufonid phological analysis of their Rhabdias fauna suggested the with a sub-Saharan distribution, the parasites being either presence of a distinct and new species in each of the four assigned to a European species (Moravec et al. 1987), or hosts. In this paper, we give a morphological description left unnamed (Saad et al. 2009). A fifth Rhabdias spe- of the new taxa and provide a molecular characterisation cies (Baylis 1929) is a parasite of Gymnophiona, a small based on the 12S rDNA and mitochondrial cytochrome c group of lissamphibians. oxidase subunit 1 (coxI) genes of three of the four species.

Address for correspondence: O. Bain, Muséum National d’Histoire Naturelle, UMR 7205 CNRS, Origine, Structure et Evolution de la Biodiversité, 61 rue Buffon, CP52, 75231 Paris cedex 05, France. Phone: +33 (0)1 40793497; Fax: +33 (0)1 40793499; E-mail: [email protected]

47 In order to assess the mode of reproduction, the genital der a stereomicroscope. Subsequently, a part of its mid-section tracts of the parasitic females of each species were ex- was removed and fixed directly in absolute ethanol for molecu- amined as in previous studies (Lhermitte-Vallarino and lar analysis. Both the anterior and posterior parts were fixed in Bain 2004, Lhermitte-Vallarino et al. 2008, 2009a, b). hot 70% ethanol. The collection and exportation of specimens Free-living stages of one of the species were obtained had been authorized by the Cameroon Ministry of Forests and through faecal culture and their development and mor- Fauna. Hosts from Cameroon and Madagascar are kept in the National Collection of the Muséum National d’Histoire Na- phology described. The aim of this study was not only to turelle (MNHN), Paris. Anurans from Cameroon were identified estimate Rhabdias diversity among lissamphibians in the by A.M. Ohler, MNHN. Afrotropical region, but to collect additional information Morphological analysis. Specimens were cleared in lac- on species from this host group to elucidate their relation- tophenol and observed under a compound microscope equipped ship with parasites from chamaeleonids. with a drawing tube. Measurements were taken from drawings and are given in micrometres unless otherwise stated. Width of Materials and methods buccal capsule corresponds to its maximum external diameter, and buccal capsule length excludes the more or less distinct The hosts. The nomenclature of amphibian hosts follows granulous posterior segment as described in Lhermitte-Vallarino that of Frost (2009). Frost et al. (2006) placed former members of the genus Bufo Laurenti (Bufonidae) in several genera, et al. (2009b). Based on previous studies (Tkach et al. 2006, amongst others Bufo, Rhinella Fitzinger, Amietophrynus and Lhermitte-Vallarino et al. 2008, Martínez-Salazar 2008), four Duttaphrynus, the latter two named by Frost et al. (2006); at ratios are provided: percentage of oesophagus length and tail the same time, several species were synonymized. In order to length of total body length (TBL), distance from apex to vulva facilitate comparison with existing literature reports, the new as percentage of TBL, and buccal capsule ratio (buccal capsule and previous names are given here, as well as in the taxonomic length/buccal capsule width). Two measurements regarding discussions. Bufo americanus Holbrook is now listed as Anax- body width are given, the first indicating body width excluding yrus americanus (Holbrook), Bu. garmani Meek, Bu. gutturalis the vesicle followed by the one including it in parentheses. Pre- Power, Bu. regularis Reuss and Bu. maculatus Hallowell have sented in the text is the type-series range of measurements for been placed into the genus Amietophrynus, and Bu. melanos- each morphological character. Individual measurements of the tictus Schneider is now listed as Du. melanostictus (Schneider) holotype and paratypes are listed in Tables 1–3. Ovaries were (Frost 2009). Kloss (1971) recorded ‘Bufo typhonius (L.)’ from examined for the presence of a male gamete production area, Belém, Pará, Brazil, as the type host of R. androgyna Kloss, named testis zone, according to Runey et al. (1978). 1971, but Frost et al. (2006) have subsequently split the Bu. Molecular analysis. Specimens analysed in this study were typhonius complex into several genera and species. Given the stored in accordance with procedures specified in the Bioreposi- geographic distribution of Kloss’ (1971) specimens, it would tories initiative (http://www.biorepositories.org) and belong to seem most likely that his material belonged to what is currently the collection identified as ‘zpl’ of Milano Bicocca institution known as Rh. margaritifera (Laurenti), a common South Ameri- (MIB). For molecular analysis freshly recovered worms were can toad occurring throughout Amazonian South America and transferred directly into absolute ethanol and kept refrigerated eastern Panama (Frost 2009). Similarly, Travassos (1926) de- at 4°C: one paratype, and mid-section of another paratype 52 scribed R. fülleborni Travassos, 1926 from ‘Bufo marinus L.’ YU from Am. gutturalis (specimen vouchers MIB:zpl:01516 from Brazil. However, several subspecies of the Bu. marinus and MIB:zpl:01517); two fragments of one paratype 97 NL complex have been synonymized and placed into different spe- from Bo. madagascariensis (specimen vouchers MIB:zpl:00226 cies within the genus Rhinella. Since Travassos (1926) listed and MIB:zpl:00227), two fragments of a second paratype 97 NL Linnaeus as host authority, it appears most likely that he was (specimen voucher MIB:zpl:00228 and MIB:zpl:00229) and an referring to a host now listed as Rh. marina (Linnaeus) by Frost additional female 99 NL (specimen voucher MIB:zpl:00230); (2009). To avoid confusion, host genera are abbreviated using mid-section of holotype 74 NL from As. rheophilus (speci- the first two letters of the generic name. men voucher MIB:zpl:00190). DNA extracts were prepared In September 2008, the lungs of five Am. gutturalis from using 5 PRIME, ArchivePure DNA Purification Kit. Rhabdias South Africa were examined for the presence of Rhabdias by species coxI amplification and sequencing were obtained fol- one of the authors (K.J.; no permit required). Several worms lowing Folmer et al. (1994) using the primer pair LCO1490 were recovered, fixed and stored in absolute ethanol. Two Boo- (5’-GGTCAACAAATCATAAAGATATTGG-3’) and HCO2198 phis madagascariensis (Peters) (Mantellidae), imports from (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’); 12S rDNA Madagascar, were necropsied; worms were fixed in hot 70% sequences were generated following Casiraghi et al. (2004) us- ethanol, except for two that were transferred directly into abso- ing the primer pair 12SF (5’-GTTCCAGAATAATCGGCTA-3’) lute ethanol for molecular analysis. and 12SdegR (5’-ATTGACGGATGRTTTGTACC-3’). PCRs The remaining material was collected in Cameroon by were performed in a volume of 20 µl under the following final

N. L.‑V. and I.I., while collaborating on a biodiversity project conditions: 1X buffer including 2.5 mM MgCl2 (MasterTaq kit, in this region (Lhermitte-Vallarino et al. 2008). Two arthroleptid EppendorfTM), 0.2 mM of each dNTP, 1 µM of each forward and species were captured on two distinct mountains in the Cam- reverse primers and 1U of DNA polymerase (MasterTaq kit, Ep- eroonese volcanic chain, a single Leptopelis brevirostris (Wern- pendorfTM). The amplicons obtained are approximately 600 and er) from Mount Cameroon and a single Astylosternus rheophilus 500 bp long, respectively. PCR products were gel-purified using Amiet from Mount Oku. Each frog harboured a single lung the 5 PRIME, GelElute Extraction Kit and sequenced directly worm. The specimen from As. rheophilus was drawn alive un- using ABI technology. Sequences were checked by eye with

48 Junker et al.: Rhabdias from Afrotropical anurans

BioEdit sequence alignment editor (version 7.0.5; Hall 1999) sule convex, and slightly serrated in some specimens using GenBank sequences as reference sequences (FM179476– (Fig. 3A); outer edge with more or less distinct internal FM179479; FN395318–FN395323) and unambiguously aligned refringent zone; anterior apex meets velum in a pointed using ClustalX (Thompson et al. 1997). The Rhabdias species tip (Fig. 3A, B). Oesophagus without shoulders, its an- sequences are deposited in the EMBL Data Library under the terior edge rarely reaching mid-level of buccal capsule. following accession numbers: R. picardiae sp. n. 12S rDNA FN434093–FN434094, coxI FN434095–FN434096; R. venc- Oesophagus 690–790 long (8.6–9.9% of TBL), slender, esi sp. n. 12S rDNA FN434097–FN434101, coxI FN434102– of equal width along anterior 2/3 of its length, then gradu- FN434106; R. tanyai sp. n. 12S rDNA FN434097–FN434101, ally widening into slim bulb; width at oesophagus mid- coxI FN434102–FN434106. The alignments were analysed length 60–70, bulb 95–130 wide (Fig. 1B). Body width using distance matrix method (i.e. neighbour joining). Nucle- at bulb 160(180)–300(360). Nerve ring observed in two otide distances have been calculated using MEGA 4.0 (Tamura specimens, at 235 and 250 from anterior extremity. Excre- et al. 2007) – options: nucleotide, Kimura 2-parameter (K2P), tory pore not identified, obscured by cells of lateral chord complete deletion, standard error computation by bootstrapping (Fig. 1B). Apex of intestine wider than bulb at oesopha- 500 replicates. geal-intestinal junction; intestine occupying entire width Rhabdias biology. For life-cycle studies of R. vencesi, four of specimen anteriorly (Fig. 1A); often filled with dark petri dish cultures were set up as detailed in Lhermitte-Vallarino and Bain (2004), using material collected from Bo. madagas- contents. cariensis (99 NL) and distilled water with active charcoal as Genital tract amphidelphic; genital bend in ovaries culture medium. Cultures were kept at room temperature and (Fig. 1C, F); anterior genital bend at 1375–2150 from an- checked regularly for developing stages for several days. Two terior extremity, posterior genital bend at 490–600 from cultures were made from eggs released from a female in vitro, posterior extremity. Oviducts and proximal end of ova- one from larvae recovered from the lungs and one from faecal ries folded upon themselves. In a single specimen, uterus larvae extracted from the rectum of the host. Only the latter obscuring posterior genital bend and extending posteriad yielded developing worms. past anus, ending at 265 from tip of tail. Male gametes with light-reflecting nuclei seen in anterior and/or poste- RESULTS rior oviduct of several specimens (Fig. 1F). Vulva without Rhabdiasidae Railliet, 1916 salient lips, slit-like, at 4535–5050 from head (55–62% of TBL). Uterus thin-walled, filled with numerous eggs, Rhabdias picardiae sp. n. Junker, Lhermitte-Vallarino most of which contain first-stage larvae. Mature eggs et Bain Figs. 1A–F, 3A–F, Table 1 100–120 long and 60–69 wide (n = 10). Small worms with variable dorsal bend. Body 8.0–8.35 Tail 270–350 long, reaching 3.5–3.8% of TBL, taper- mm long, 460(530)–580(600) wide at mid-length. Cu- ing quickly to rounded tip. Body width at anus 120(240)– ticular vesicle with irregular folds and indentations along 225(440). Caudal vesicle voluminous with irregular in- entire length of body, including tip of tail (Fig. 1C, D); dentations; terminal, elliptical to lance-shaped segment in median view, vesicle attached laterally to inner layer surrounding tip of tail. Tip of tail with patch of fine granu- of cuticle via conspicuous fibres associated with subcu- lar material in 5/7 specimens (Fig. 1D). ticular pores (Fig. 1B); at anterior extremity more or less pronounced inflation of cuticle extending posteriorly to T y p e h o s t : Amietophrynus gutturalis (Power) (= Bufo gutturalis Power) (Bufonidae), 52 YU. approximately level of oesophageal-intestinal junction; vesicle less conspicuous in mid-section and again more T y p e l o c a l i t y : Pretoria (25.66S, 28.15E), Gauteng Prov- ince, South Africa. Collection date: 01 September 2008. pronounced in tail-region. Lateral chords thick, filling S i t e o f i n f e c t i o n : Lungs. body cavity in anterior region (Fig. 1B, E). Head with six papillae in apical view (Fig. 3C, D). P r e v a l e n c e a n d i n t e n s i t y : 2/5 hosts infected with five (52 YU) and three (53 YU) parasites, respectively. Four submedian papillae arranged in square, anterior to lemon-shaped oral opening (Fig. 3D); each papilla with T y p e m a t e r i a l : 52YU. Deposited in the MNHN collection: holotype female, one entire paratype female, two paratype salient sensillum and slightly protruding base. Two very females with anterior and posterior parts. One paratype fe- small lateral papillae posterior to submedian papillae lev- male and mid-section of another used for molecular analysis; el. Amphidian canals barely visible. Cuticular velum lim- specimen vouchers MIB:zpl:01516 and MIB:zpl:01517. One iting oral opening, its lumenal edge drawn out into a sharp paratype (N-938) in the Institute of Parasitology, BC ASCR, tip in longitudinal section (Fig. 3A, B). České Budějovice, Czech Republic. EMBL Data Library ac- Lumen of buccal cavity round in transverse section cession numbers: 12S rDNA FN434093–FN434094, coxI (Fig. 3E), triangular at oesophageal junction. Vestibu- FN434095–FN434096. lum short, 5–7 long. Buccal capsule wall triangular in A d d i t i o n a l m a t e r i a l : 53YU. Deposited in the MNHN longitudinal section, 7–10 long and 23–25 wide, with collection. granulous posterior segment where it joins oesophagus; E t y m o l o g y : The species is named after Dr. Jackie A. Picard, buccal capsule ratio 0.3–0.4; inner edge of buccal cap- who kindly made the hosts available to us.

49 Table 1. Morphometric characters of Rhabdias picardiae sp. n. from Amietophrynus gutturalis (Power) (Bufonidae) in South Africa. All measurements in micrometres unless otherwise indicated.

Holotype Paratypes MNHN collection 52 YU 52 YU 52 YU 52 YU 53 YU 53 YU 53 YU Specimen 2 1 3 4 1 2 3 Length (mm) 8.15 8.0 – – 8.0 8.35 – Width at mid-body (with vesicle) 460 (530) 470 (515) – – 510 (580) 580 (600) – Vestibulum depth 7 5 6 – 6 5 6 Buccal capsule length 10 8 10 9 7 8 8 Buccal capsule max. external diameter 23 24 23 25 24 23 24 Buccal capsule ratio 0.4 0.3 0.4 0.4 0.3 0.3 0.3 Oesophagus length 720 700 710 690 790 720 – Oesophagus width at mid-length 60 60 60 70 60 70 – Bulb diameter 105 95 100 95 120 130 115 Body width at bulb (with vesicle) 170 (195) 160 (180) 280 (325) 265 (300) 205 (320) 300 (360) 240 (310) Head to vulva 5050 4900 – – 4535 4575 – Anterior genital bend – head 2150 2000 – 1700 2075 1375 – Posterior genital bend – tip of tail 600 540 490 – 600 265a – Tail length 300 280 270 350 300 300 – Width at anus (with vesicle) 120 (240) 120 (290) 225 (440) 170 (220) 160 (250) 170 (250) – Apex to nerve ring – – 250 – 235 – – Oesophagus length as % of body length 8.8 8.8 – – 9.9 8.6 – Head to vulva as % of body length 62.0 61.3 – – 56.7 55.0 – Tail length as % of body length 3.7 3.5 – – 3.8 3.6 –

aUterus obscures posterior genital bend (pgb) and extends further posteriad than pgb.

Remarks. The first species to be compared with the al. 1987) vs. 9–12 long and 8–10 wide in European speci- present material are the four Rhabdias recorded from mens (Hartwich 1972). Rhabdias bufonis sensu Moravec, Afrotropical anurans (Table 3): R. africanus Kuzmin, Baruš et Ryšavý, 1987 has a longer buccal capsule than 2001, type host Am. maculatus (Hallowell) (= Bu. macu- R. picardiae, a shorter oesophagus (510 vs. 690–790) and latus), South Africa; R. bufonis (Schrank, 1788), assigned a longer body (13.02 mm vs. 8.35 mm). by Moravec et al. (1987) to lung worms recovered from Rhabdias collaris has similar structures attaching the Am. regularis (Reuss) (= Bu. regularis) in Egypt; R. col- vesicle to the body, but its head is conspicuously thick- laris Baker, 1987, type host Le. vermiculatus (Boulanger) ened by peripheral muscle bundles, its oesophagus is (Arthroleptidae) in Tanzania; and R. madagascariensis shorter and shaped differently (Baker 1987b). Chabaud, Brygoo et Petter, 1961, type host Ptychadena Rhabdias madagascariensis is shorter, 3.55 mm long, mascareniensis (Duméril et Bibron, 1841) [= Rana (Pty- the oesophagus is club-shaped with a distinct dilatation chadena) mascareniensis] (Ptychadenidae) in Madagas- anterior to nerve ring and the intestinal apex is narrower car. than the base of the oesophagus (Chabaud et al. 1961). Rhabdias africanus has a longer body (12.45–19.8 Rhabdias bdellophis Baylis, 1929 was described vs. 8–8.35), relatively shorter oesophagus (3.5–4.7% vs. from the caeciliid Scolecomorphus vittatus (Boulanger) 8.6–9.9%), twice longer buccal capsule (15–20 vs. 7–10) (= Bdellophis vittatus) (Gymnophiona), in Tanzania. It is and two lateral pseudolabia (Kuzmin 2001), which we, distinct from R. picardiae in the “very vaguely defined however, consider simple swellings at the level of the am- buccal capsule”, the about three times longer tail and phids. slightly larger body (Baylis 1929) (Table 3). Rhabdias bufonis is in the following referred to as Compared to the Rhabdias species from anuran hosts R. bufonis sensu Moravec, Baruš et Ryšavý, 1987, be- world-wide, R. picardiae is close to R. americanus Bak- cause the parasites from Am. regularis, a host with a strict- er, 1978 from Anaxyrus americanus (Holbrook) (= Bu. ly Afrotropical distribution (Frost 2009), differ from the americanus) (Bufonidae) in Canada in that the oesopha- redescriptions of Travassos (1930) and Hartwich (1972). gus is 770–870 long with a slightly inflated bulb, and its The latter had been based on specimens recovered from tail is entirely enclosed in the cuticular vesicle (Baker European bufonids and ranids, as was the original de- 1978, Kuzmin et al. 2003). However, R. americanus is scription (not available). The main distinctive character is distinct in having a thin tail (length/width ratio about 6 vs. the size of the buccal capsule, which is of diagnostic im- 1–2.25), longer body (10.7–14.1 mm), well-developed portance (Lhermitte-Vallarino and Bain 2004, Lhermitte- lateral pseudolabia, a narrower buccal cavity (12–15 vs. Vallarino et al. 2008); irrespective of body size, it is 15 23–25), a much deeper vestibulum and a thicker vesicle long and 21 wide in specimens from Egypt (Moravec et (Baker 1978).

50 Junker et al.: Rhabdias from Afrotropical anurans

Fig. 1. A–F. Rhabdias picardiae sp. n. from Amietophrynus gutturalis in South Africa. A – habitus, left lateral view, holotype; B – anterior region, median view; C – posterior region, left lateral view; D – caudal extremity; E – transverse section at level of oesophagus, half body representation showing one large lateral chord; F – bend of terminal ovary and a part of oviduct with spermatozoa in the lumen, remainder of oviduct tightly coiled and hidden behind ovary. G–M. Rhabdias vencesi sp. n. from Boophis madagascariensis in Madagascar. G – anterior region, median view, with proximal part of excretory glands; H – posterior region, left lateral view; I – anterior region of another specimen, before fixation, with excretory glands and pore, lateral right view;J – end of posterior ovary, oviduct with ovulae and spermatozoa-like cells, and beginning of uterus; K – habitus, left lateral view; L, M – head of two females, more or less invaginated. Scale bars: A, body length 8.15 mm; B, D, G = 100 µm; C, H, I, J = 200 µm; E, F, L, M = 70 µm; K, body length 12 mm.

51 Table 2. Morphometric characters of Rhabdias vencesi sp. n. from Boophis madagascariensis (Peters) (Mantellidae) in Madagascar. All measurements in micrometres unless otherwise indicated.

Holotype Paratypes Additional material MNHN collection 97 NL 97 NL 97 NL 97 NL 97 NL 97 NL 97 NL 97 NL 97 NL 99 NL 99 NL 99 NL Specimen 8 1 2 3 4 5 6 7 9 1 2 3 Length (mm) 12.0 11.4 13.2 12.1 13.1 11.8 9.9 11.3 – 9.5 10.5 9.3 Width at mid-body (with vesicle) 600 680 620 600 700 770 680 640 – 570 630 560 (630) (810) (700) (600) (650) (580) Vestibulum depth 6 10 – 6 – 6 – 5 – 10 12 6 Buccal capsule length 6 10 – 9 6 6 7 7 6 6 6 5 Buccal capsule max. external diameter 20 20 – 22 20 19 20 21 20 22 22 19 Buccal capsule ratio 0.3 0.5 0.4 0.3 0.3 0.3 0.4 0.3 0.3 0.3 0.3 0.3 Oesophagus length 525 550 520 550 550 570 540 530 550 500 495 540 Oesophagus width at mid-length 80 80 – 80 80 85 80 80 80 72 72 80 Bulb diameter 120 120 110 100 120 110 120 120 120 100 95 110 Body width at bulb (with vesicle) 270 230 210 250 270 300 260 250 340 240 250 220 (290) (240) (280) (290) (325) (280) (270) (370) (280) (300) (250) Head to vulva 6150 6400 6400 6490 6650 6000 4850 5500 – 5050 5350 4600 Anterior genital bend – head 1150 620 1000 1130 940 1030 650 930 1100 930 1250 620 Posterior genital bend – tip of tail 1130 – 1130 1070 – 1060 1080 900 – 700 1075 1100 Tail length 340 315 320 260 300 305 350 290 – 230 375 240 Width at anus (with vesicle) 140 140 120 140 150 110 150 130 – 120 135 100 (180) (320) (170) (160) (170) (140) (220) (155) (155) (145) (130) Apex to nerve ring 200 200 – 185 190 180 180 205 – 195 210 190 Oesophagus length as % of body length 4.4 4.8 4.0 4.5 4.2 4.8 5.5 4.7 – 5.3 4.7 5.8 Head to vulva as % of body length 51.5 56.0 48.7 53.7 50.8 50.8 49.2 48.7 – 53.2 51 49.5 Tail length as % of body length 2.8 2.8 2.4 2.1 2.3 2.6 3.5 2.6 – 2.4 3.6 2.6

Rhabdias vencesi sp. n. Junker, Lhermitte-Vallarino et at 180–210 from head. Excretory pore observed in one Bain Figs. 1G–M, 3G–K, Table 2 specimen before fixation (Fig.1I); anterior part of excretory glands identified (Fig. 1G); at 205 from anterior Body more or less straight or slightly bent dorsal- extremity. Apex of intestine bell-shaped, narrow at level ly, 9.5–13.2 mm long. Width at mid-body 560(580)– of oesophageal-intestinal junction, but rapidly widening 770(810). Cuticle: vesicle more conspicuous in cephalic posteriorly to fill almost entire width of body (Fig. 1I); region, with internal transverse striae that are spaced at anterior intestinal wall thick, with conspicuous, densely irregular intervals of approximately 20–30 and 10–20 in crowded, large pear-shaped cells (Fig. 1G); cells take on two specimens measured; thin or indistinct along body, a more conventional shape at approximately level of ante- but slightly more prominent in tail region. rior genital bend. Posterior part of intestine usually filled Head with six papillae in apical view; four large, mam- with dark brown contents. milate submedian and two smaller lateral papillae, each Genital tract amphidelphic, posterior and anterior with terminal transparent cuticular apex and sensory sen- genital bend at level of muscular oviduct (Fig. 1H, J); sillum; lateral papillae slightly posterior, submedian pa- anterior genital bend at 620–1250 from head; posterior pillae directly adjacent to oral opening; amphids open on genital bend at 700–1130 from tip of tail. Oviducts and external surface of lateral papillae (Fig. 3H, I). Oral open- proximal end of ovaries folded upon themselves. Numer- ing dorso-ventrally flattened, oval to spindle-shaped; ve- ous small, globular cells, identified as male gametes, seen lum absent. Vestibulum 5–12 long, difficult to discern in in lumen of anterior oviduct of five specimens, in which specimens with more or less invaginated head, resulting this character was studied (Fig. 1J); unusual non-light re- in wide range of measurements. Buccal capsule obscured flecting nuclei of spermatozoa suggest they are degener- by thickened apex in some invaginated specimens; no ating; same cells present in posterior oviduct of one of posterior segment identified; length 5–10, width 19–22, these specimens, but posterior oviduct of remaining four roughly triangular in longitudinal section, round in api- specimens obscured by intestinal contents. Vulva slit-like, cal view; lumen cylindrical. Buccal capsule ratio 0.3–0.5. inconspicuous, without salient lips, situated in mid-region Oesophagus with shoulders, its apex extending anteriorly of body at 4600–6650 from head or 48.7–56% of TBL. to mid-level or apex of buccal capsule; stout, club-shaped, Apex of ovaries far overlapping level of vulva. Uterus 495–570 long, reaching 4.0–5.8% of TBL (Fig. 1G). sac-like, thin-walled, filled with numerous eggs, the ma- Oesophagus width at mid-length 72–85, gradually wid- jority of which contain first-stage larvae. Mature eggs ening to maximum width of 95–120 at bulb. Body-width 110–148 long and 56–72 wide (n = 10). at bulb level 210(240)–340(370). Slight dilatation of Tail conical, tapering gradually to slightly rounded tip. oesophagus immediately prior to nerve ring. Nerve ring Tail length 230–375 or 2.1–3.6% of TBL. Caudal vesi-

52 Junker et al.: Rhabdias from Afrotropical anurans cle with conspicuous inflation at anus, body width at that developed buccal capsule and a relatively longer oesopha- level 100(130)–150(220). Tip of tail without vesicle. gus (7.8–8.0 vs. 4.0–5.8% of tbl). When compared to Rhabdias species from anuran hosts T y p e h o s t : Boophis madagascariensis (Peters) (Mantellidae). world-wide, an arrangement of head papillae with well- T y p e l o c a l i t y : Import from Madagascar; exact locality developed, salient lateral papillae as seen in R. vencesi is unknown. Collection date: 28 February 2008. also present in R. joaquinensis Ingles, 1935 from a ranid S i t e o f i n f e c t i o n : Lungs. in California, Rana aurora Baird et Girard (Kuzmin et P r e v a l e n c e a n d i n t e n s i t y : Type host specimen al. 2003). This species is distinct, however, in having an (97NL) harbouring 11 females, and a second host (99NL) attenuated anterior body half, a postequatorial vulva and harbouring 4 females. a cylindrical oesophagus (Kuzmin et al. 2003). T y p e m a t e r i a l : 97NL. Deposited in the MNHN collection: female holotype, eight paratype females. Two fragments of two paratypes each used for molecular analysis; specimen Rhabdias ohlerae sp. n. Junker, Lhermitte-Vallarino et vouchers MIB:zpl:00226, MIB:zpl:00227, MIB:zpl:00228 Bain Figs. 2A–F, 3L, Q, Table 3 and MIB:zpl:00229 respectively. One paratype (N-939) in Body small, 6.5 mm long, dorsally bent, tapering at the Institute of Parasitology, BC ASCR, České Budějovice, both ends; width at mid-body 380(420). Thin vesicle Czech Republic. EMBL Data Library accession numbers: 12S present along entire length of body, including tip of tail; rDNA FN434097–FN434101, coxI FN434102–FN434106. difficult to discern in some places. Anterior extremity ta- A d d i t i o n a l m a t e r i a l : 99NL. Deposited in the MNHN pering rapidly from level of anterior genital bend anteriad, collection: three females. One female used for molecular terminating in distinct dilatation that is set off from body analysis (specimen voucher MIB:zpl:00230). by slight constriction (Fig. 2D, C); cephalic dilatation 90 E t y m o l o g y : The new species is named after Dr. Miguel long, 70 wide. Mouth opening limited by cuticular ve- Vences in recognition of his significant contribution to our lum. Vestibulum 5 deep. Buccal capsule 11 long and 16 knowledge of the amphibian fauna of Madagascar. wide; ratio 0.7; posterior segment forming distinct ridges, Remarks. Rhabdias vencesi most closely resembles where it rests upon oesophagus (Fig. 3L); lumen of buc- R. madagascariensis. In both species the oesophagus is cal capsule funnel-shaped. Oesophagus club-shaped, 450 club-shaped with an anterior dilatation prior to the nerve long (6.9% of TBL), with shoulders. Apex of oesophagus ring, and the apex of the intestine is narrow. However, not reaching anterior border of buccal capsule (Fig. 3L). R. madagascariensis is readily distinguished by its small- Anterior dilatation of oesophagus present at 100 from er body and the proportionally longer oesophagus (3.55 head, 41 wide, anterior to nerve ring (Fig. 2D). Subse- vs. 9.3–13.2 mm, and 7.6 vs. 4.0–5.8% of TBL) and quently, gradual increase in oesophagus width from 35 at the anterior part of the intestine, which remains slender mid-length to maximum of 55 at indistinct posterior bulb. (Chabaud et al. 1961), whereas that of R. vencesi expands Nerve ring and excretory pore at 150 and 190 from apex, rapidly. Its mouth is round as opposed to dorso-ventrally respectively. Apex of intestine with a shallow depression flattened as inR. vencesi. to accommodate base of oesophagus, which is narrower The remaining Afrotropical species are equally distinct than intestinal apex at junction (Fig. 2D). Intestine thick- (Table 3). Rhabdias africanus differs in having two dis- walled, its cells forming a distinct giraffe pattern with nar- tinct swellings at the level of the amphids, papillae that row interstitial spaces (Fig. 2D); taking up entire width overhang the oral opening giving the mouth an octago- of body anteriorly, but narrowing at approximately level nal shape, a longer buccal capsule (15–20 vs. 5–10) and of anterior genital bend; filled with dark brown contents a slender oesophageal bulb (65–80 vs. 95–120) (Kuzmin posteriorly. 2001). While being of similar length (9–12 vs. 5–10), the Genital tract amphidelphic. Anterior genital bend in buccal capsule ratio of R. bufonis sensu Moravec, Baruš ovary, 850 from head; posterior genital bend in oviduct et Ryšavý, 1987 is greater than that of R. vencesi (0.7 (Fig. 2F), 675 from tip of tail. Oviducts and proximal vs. 0.3–0.5) and the intestinal apex is broader than the end of ovaries folded upon themselves. Ovary apices just oesophageal base (Moravec et al. 1987). Rhabdias col- overlapping vulva. Narrow band (30–50 wide) of small, laris has a conspicuous cephalic swelling, a round mouth poorly defined and irregularly arranged cells in posterior and a distinctly longer vestibulum (42 vs. 5–12) (Baker ovary interpreted as testis zone (Fig. 2E). Vulva without 1987b). Rhabdias picardiae is shorter, has a lemon- salient lips, at 3120 from head (48% of TBL). Uterus shaped mouth opening, small lateral papillae and a buccal thin-walled, sac-like, filled with numerous eggs, many capsule with a distinct posterior segment. Furthermore, its of which contain first-stage larvae. Mature eggs 104–120 oesophagus is long and narrow (8.6–9.9 vs. 4.0–5.8% of long by 58–65 wide (n = 10). TBL) and the intestinal apex is wider than the oesophagus Tail conical, 275 long, reaching 4.2% of TBL. Width at the oesophageal-intestinal junction. Rhabdias bdello- at anus 140 (190). Distinct postanal swelling of body in phis is distinct in having a shorter body and tail, a weakly lateral view (Fig. 2B). Caudal vesicle slightly more con-

53 Fig. 2. A–F. Rhabdias ohlerae sp. n. from Leptopelis brevirostris in Cameroon. A – habitus, left lateral view; B – posterior region, left lateral view; C – head, lateral view; D – anterior region, left lateral view; E – proximal part of male area in ovary (top of drawing indicates direction of ovary apex); F – end of posterior ovary, oviduct and beginning of uterus. G–M. Rhabdias tanyai sp. n. from Astylosternus rheophilus in Cameroon. G – anterior region, before fixation;H – oviduct with ovulae and, possibly degenerate, spermatozoa up to beginning of uterus; I – oesophageal region with one of the excretory cells, dorso-ventral view; J – apex of intestine; K – posterior region with end of intestine and rectum, ventro-dorsal view (anus not identified); L – caudal extremity, ventro- dorsal view; M – anterior extremity, before fixation. Scale bars: A, body length 6.5 mm; B, D, F, H, I, J = 200 µm; C, E, L = 50 µm; G = 500 µm; K = 300 µm; M = 60 µm.

54 Junker et al.: Rhabdias from Afrotropical anurans spicuous than along body, with irregular folds. Tip of tail Rhabdias tanyai sp. n. Junker, Lhermitte-Vallarino et 50 long, demarcated from rest of tail by sharp bend, en- Bain Figs. 2G–M, 3M, Table 3 closed in thin vesicle. Body with slight dorsal bend (Table 3), length >13.9 mm Type host: Leptopelis brevirostris (Werner) (Arthrolepti- (length of anterior plus posterior fragment). Thin vesicle dae). present along entire length of body, including tip of tail. T y p e l o c a l i t y : Petit Mont, Etinde Summit, Mount Cam- Buccal capsule and oesophageal apex forming a flat- eroon, Cameroon (4.06N, 9.12E). Collection date: 28 April topped projection at cephalic end in fixed specimen; the 2007. same projection observed prior to fixation, but less pro- S i t e o f i n f e c t i o n : Lungs. nounced (Figs. 2 G, I, M, 3M). Buccal capsule 12 long P r e v a l e n c e a n d i n t e n s i t y : A single specimen from and 23 wide, with a ratio of 0.5; posterior segment not a single host. resting on but rather inserted into the oesophageal apices T y p e m a t e r i a l : Holotype female: 26NL. Deposited in the (Fig. 3M); anterior and posterior segments form shoe sole- MNHN collection. shape; lumen of buccal capsule cylindrical. Oesophagus E t y m o l o g y : The species is named after Prof. Anne-Marie club-shaped, with shoulders, but apex not reaching mid- Ohler, for her contribution to the taxonomy of Asiatic am- level of buccal capsule; 650 long, reaching a maximum phibians and her assistance with the identification of some of of 4.7% of TBL when taking TBL as the sum of the an- the hosts examined herein. terior and posterior fragment; oesophagus width at mid- Remarks. The distinct cephalic dilatation readily length 60, gradually increasing to maximum of 95 at bulb. distinguishes R. ohlerae from its congeners in the Afro- Body width at bulb 270 (290). Nerve ring at 235 from tropical region, excepting R. collaris, as well as from the apex, excretory pore not observed, but a long excretory majority of Rhabdias species from anuran hosts world- gland identified. Apex of intestine bell-shaped, narrow at wide. While in R. collaris the cephalic inflation consists oesophageal-intestinal junction, but rapidly widening to of two lobe-like, divergent structures, which open a wide, take up almost entire width of body (Fig. 2G). Intestine funnel-shaped vestibulum of considerable depth between thick walled, its cells arranged in a giraffe pattern with them (42, measured on drawing) (Baker 1987b), that of wide interstitial spaces (Fig. 2J); filled with dark brown R. ohlerae is round, with a small oral opening that leads contents posteriorly; terminating in two short, peg-like into a short vestibulum (5 deep). extensions. From anurans world-wide, the two other species with Genital tract amphidelphic. Removal of mid-section cephalic dilatations that are not based on vesicular infla- made measurements impossible. Vulva absent from both tion only are R. androgyna Kloss, 1971 from a Brazil- fragments. It is difficult to assess if the following obser- ian host identified as Bu. typhonius (L.) by Kloss (1971), vations are typical features or result from specimen ma- but most likely Rh. margaritifera (Laurenti) (Frost 2009), nipulation. Anterior oviduct-uterus junction observed, and R. sphaerocephala Goodey, 1924 from the toad Bu. with uterus extending considerable distance anteriad from bufo (Linnaeus) (= Bu. vulgaris) in England, with addi- junction. Anterior genital bend not seen, but uterus clearly tional details on head morphology given by Kuzmin et anterior-most portion of anterior genital tract. Ovaries dis- al. (2007). Rhabdias androgyna, as illustrated by Kloss posed in generous loops across width of body, containing (1971), is distinguished from the new species by a thicker ovulae arranged in transverse bands of 5–6 each. Testis body vesicle, deep vestibulum and longer body as well as zone identified in posterior ovary. Lumen of anterior oesophagus (9–13 mm and 577–618, respectively). oviduct containing similar but smaller cells represent- Rhabdias ohlerae most closely resembles R. sphae- ing spermatozoa, possibly degenerate (Fig. 2H). Uterus rocephala, being similar in body length (6.5 mm vs. thin-walled, sac-like, filled with numerous eggs, many of 6–6.5 mm) and oesophagus length (450 or 6.9% of TBL which contain first-stage larvae. Mature eggs 96–105 long vs. 430–450 or 6.6–7.5% of TBL) (Goodey 1924a; calcu- by 50–62 wide (n = 5). lations based on minimum and maximum values). Further Tail conical, with short, stubby tip, 275 long, reach- similarities are the shape of the oesophagus, including the ing a maximum of 2.0% of TBL, when taking TBL as the anterior swelling situated just posterior to the cephalic sum of the anterior and posterior fragments. Tip of tail dilatation, and the apex of the intestine, which encom- enclosed in vesicle. passes the base of the oesophagus. However, the cephalic T y p e h o s t : Astylosternus rheophilus Amiet (Arthrolepti- dilatation of R. sphaerocephala is distinctly wider than dae). that of R. ohlerae (115–120 vs. 70; both sets of measure- T y p e l o c a l i t y : Forest below Oku village (6.22N, 10.52E), ments excluding vesicle), the vesicle is thicker and the tail Mount Oku, Cameroon. Collection date: 10 May 2007. is shorter in R. sphaerocephala (190–220 or 2.9–3.4 % of S i t e o f i n f e c t i o n : Lungs. TBL vs. 275 or 4.2 % of TBL) (Goodey 1924a; calcula- P r e v a l e n c e a n d i n t e n s i t y : A single female from tions based on minimum and maximum values). a single host.

55 Fig. 3. A–F. Rhabdias picardiae sp. n. A – head, dorso-ventral view; B – head, lateral view (another specimen); C–E – frontal view; C – head; D – sensory papillae and mouth; E – buccal capsule and apex of oesophagus, transverse optical section; F – diagrammatic representation of holotype. G–K. Rhabdias vencesi sp. n. G – head, subdorso-ventral view; H, I – frontal view; H – head; I – sensory papillae and mouth; J – buccal capsule, apex of oesophagus, and the particular lateral structures along the oesophagus, transverse optical section; K – diagrammatic representation of holotype. L. Rhabdias ohlerae sp. n., buccal cavity and capsule, lateral view. M. Rhab- dias tanyai sp. n., buccal cavity and capsule, dorso-ventral view. N–P. Free-living stages of R. vencesi sp. n. N – male, posterior region, left lateral view; O, P – infective larva, anterior and posterior region, right and left lateral view, respectively. Q. Rhabdias ohlerae sp. n., diagrammatic representation of holotype. Scale bars: A, B, D, E, G, I, L, M, P = 20 µm; C, H, J, N, O = 30 µm; F, K, Q = 3 mm.

T y p e m a t e r i a l : 74NL; a single worm recovered. Depos- Remarks. Rhabdias tanyai can be distinguished from ited in the MNHN collection: posterior and anterior part of species hitherto described from Malagasy and African female holotype. Mid-section removed for molecular analy- hosts (Table 3) by the cephalic projection, the shoe sole- sis; specimen voucher MIB:zpl:00190. EMBL Data Library shape of its buccal capsule and the way the posterior seg- accession numbers: 12S rDNA FN434097–FN434101, coxI ment of the capsule is inserted into the oesophagus. FN434102–FN434106. The only other Rhabdias species from anuran hosts E t y m o l o g y : The new species is named after Dr. Vincent with an anterior cephalic projection similar to that seen in Tanya, our colleague in Cameroon (EU projects on onchocer- R. tanyai is R. brachylaimus (von Linstow, 1903), origi- ciasis), and in recognition of his administrative help concern- nally described by von Linstow (1903) as Angiostomum ing the field trip. brachylaimus from Du. melanostictus (Schneider) (= Bu.

56 Junker et al.: Rhabdias from Afrotropical anurans – – – – – – 90 n = 2 Vague Vague vittatus 7.8–8.0 2.5–2.8 5.1–5.6 Tanzania 400–450 350–420 125–155 160–170 bdellophis Caeciliidae Baylis 1929 Scolecomorphus 1961 * * – – – – – – 52 7.6 3.5 270 150 125 130 1.5 8.5 3.55 n = 1 Ptychadena Madagascar Ptychadenidae mascareniensis madagascariensis Chabaud et al. includes very young and fully gravid females; d 5–12 5–10 19–22 72–85 97 NL 97 NL n = 12 0.3–0.5 4.0–5.8 2.1–3.6 95–120 48.7–56 Boophis 9.5–13.2 495–570 560–770 210–340 230–375 100–150 180–210 Mantellidae Madagascar vencesi sp. n. madagascariensis b d – – – – – – 15 21 0.7 57–72 Egypt n = 10 bufonis 288–510 163–476 144–420 168–240 regularis 2.99–13.02 ± equatorial Moravec et al. 1987 53 YU 53 from Afrotropical from lissamphibian hosts. Host nomenclature according to Frost 5–7 7–10 n = 7 23–25 55–62 60–70 0.3–0.4 8.6–9.9 3.5–3.8 95–130 8.0–8.35 690–790 460–580 160–300 270–350 120–225 235–250 gutturalis South Africa South Bufonidae picardiae sp. n. picardiae Amietophrynus 52 YU and 52 calculations based on measuremnets of holotype; Rhabdias c c * maculatus – – – – 16 0.7 n = 9 15–20 20–23 65–80 3.5–4.7 1.6–3.2 570–710 300–450 250–400 africanus 46.8–49.7 12.45–19.8 South Africa South sp. n. and other Kuzmin 2001 armani & g sensu Moravec et al. 1987; b c c * c * * * – – – – – 11 56 17 42 5.6 4.1 0.6 n = 6 collaris 9.1–10.2 480–540 370–430 190–245 Tanzania Baker 1987 vermiculatus Rhabdias ohlerae Leptopelis 5 11 48 16 35 55 6.5 6.9 4.2 0.7 sp. n., 450 380 125 275 140 150 74 NL 1 74 NL Holotype Cameroon brevirostris ohlerae sp. n. Arthroleptidae a a a – 4 – 12 23 60 95 0.5 650 800 270 300 235 > 13.9 26 NL 1 26 NL Holotype max. 2.2 max. 4.7 Cameroon rheophilus tanyai sp. n. Rhabdias tanyai Astylosternus able 3. Morphometric characters of calculated from drawings.

Based on body length being the sum of the anterior and posterior fragments; Specimen number Buccal capsule length Oesophagus length Length (mm) (2009). All measurements in micrometres unless otherwise indicated. (2009). T Host family MNHN collection and specimen number (this paper) or reference a * Host genus Host species Head to vulva as % of body length Buccal capsule max. external diameter Buccal capsule ratio Width at mid-body (with vesicle) Width Host locality Rhabdias species Oesophagus width at mid-length Tail length as % of body Tail Vestibulum depth Vestibulum Bulb diameter Body width at bulb (with vesicle) Tail length Tail Width at anus (with vesicle) Width Apex to nerve ring Oesophagus length as % of body

57 melanostictus) in Thailand and subsequently by Yuen discussion (1965) from lissamphibians in Malaysia. Based on von Linstow’s (1903) description, R. brachylaimus is, how- The Rhabdias fauna of Afrotropical anurans appears ever, considerably shorter than R. tanyai (8.8 mm vs. diverse and to date, including the present paper, eight >13.9 mm) and its oesophagus and tail are longer, reach- species have been reported from four families of anuran ing 6.7% and 4% of TBL, respectively. While specimens hosts (Table 3). Neither these lungworms nor their hosts of R. brachylaimus described by Yuen (1965) are compa- have been recorded from any of the other zoogeographic rable to R. tanyai in body length, their oesophagus ranges regions. from 270 to 370 in length only, compared to 650. In the new species, the shape and structure of the buccal capsule is diverse; it can comprise a single segment as Molecular analyses in R. vencesi, or two more or less distinct ones as in the Taking an integrated taxonomical approach based on remaining three species; its wall can be simple, or double both morphological characterisation and classical DNA as in R. picardiae. The mouth is subsphaerical or dorso- barcoding (Ferri et al. 2009), DNA sequences of three of ventrally flattened (R. picardiae and R. vencesi, respec- the four species described herein were analysed. Compar- tively), and the lateral head papillae may be developed or ison of the 12S rDNA and coxI sequences of R. picardiae, reduced. These characters cannot be compared with many R. tanyai and R. vencesi confirmed their validity. other species, but buccal capsule size, shape and size of Data obtained, based on K2P distance matrix, showed the oesopahgus, body size and the configuration of the a low nucleotide intraspecific distance both forR. vencesi. apex of intestine, to name a few, can be used for species (12S rDNA 0.2% and coxI 0.2%) and R. picardiae (12S differentiation (Kuzmin et al. 2003, Tkach et al. 2006). rDNA 0.3% and the sequences were identical in coxI), for The latter are equally useful when distinguishing parasites which more than a single specimen could be analysed. from chamaeleonids (Lhermitte-Vallarino and Bain 2004, Moreover, our results confirmed placement of the three Lhermitte-Vallarino et al. 2008, 2009a, b). species in three distinct Molecular Taxonomic Units At present, the Rhabdias fauna of Afrotropical anurans (MOTU). In fact, the nucleotide interspecific distances presents the following picture. Three species, R. bufonis (K2P) for the two molecular markers 12S rDNA and coxI, sensu Moravec, Baruš et Ryšavý, 1987, R. africanus, and respectively, are: 6.7% (standard error 1.4%) and 9.9% R. picardiae, parasitise hosts of the family Bufonidae, (standard error 1.7%) between R. picardiae and R. venc- represented by a single genus, Amietophrynus (formerly esi; 5.8% (standard error 1.4%) and 13.2% (standard er- included in Bufo), which is limited to sub-Saharan Af- ror 1.2%) between R. picardiae and R. tanyai; and 7.9% rica (Frost 2009). All four host species occur in savanna (standard error 1.6%) and 16.2% (standard error 1.7%) habitat and their distribution overlaps to varying degrees, between R. vencesi and R. tanyai. with Am. gutturalis having the widest and Am. maculatus the narrowest geographic range (Passmore and Carruthers Free-living stages of Rhabdias vencesi 1979). Several comments can be made when considering Descriptions of free-living stages of R. vencesi are the Rhabdias species and host families in the Afrotropics. based on material obtained from cultured larvae collected In bufonids, while R. picardiae and R. bufonis sensu from the rectum of host 99 NL. Male and female adults Moravec, Baruš et Ryšavý, 1987 are distinctly different were observed after 3 days, and several motile infective (Table 3), their hosts are morphologically very close. Mo- larvae were detected 7 days after the culture was started. lecular evidence, however, suggests that the two bufonids Soon a high mortality rate was observed due to bacterial separated some 5 to 6 million years ago (Channing and colonies. Howell 2006). The two South African species, R. picar- Female: not measured, not drawn. Male (n = 1) diae and R. africanus, are morphologically no closer to (Fig. 3N): body length 578, with short lateral alae; tail 53 each other than they are to R. bufonis sensu Moravec, long, including a tail filament of 9 length; spicules 26 and Baruš et Ryšavý, 1987. In fact, with respect to relative 28, gubernaculum 13 long. Caudal papillae not studied in oesophagus length, R. africanus and R. picardiae occupy detail, but two subterminal pairs conspicuous. opposite extremes of the wide range seen in this character Infective third-stage larva (n = 1) (Fig. 3O, P): en- in anuran hosts. The host of R. picardiae was collected in closed in exuvial sheath, with checkered ornamentation; the Central Bushveld bioregion, whereas the two hosts of posterior part of sheath regularly attenuated, without R. africanus were collected in the South African Lowveld subterminal indentation and bosses; tip of sheath blunt. Bushveld in the Kruger National Park (Mucina and Ru- Tail of larva tapering to conical, sharply pointed tip. therford 2006). However, the three species from African Measurements exclude sheath: 575 long, 20 wide; nerve bufonids share a common trait regarding oesophagus ring at 80 from head; buccal cavity funnel-shaped; buc- morphology: the bulb is not very distinct, and the anterior cal capsule 11 deep, 7 wide, composed of two segments; dilatation, which is relatively common in Rhabdias from oesophagus 175 long; tail 58 long; genital anlage 50 long, other bufonids (Goodey 1924a, Yuen 1965, Kloss 1971, situated at mid-length of intestine. Tkach et al. 2006, Martínez-Salazar and León-Règagnon

58 Junker et al.: Rhabdias from Afrotropical anurans

2007), is absent. In addition, none of the species from narrower (≤17) than that of the new taxa described herein African bufonids have well-developed pseudolabia. Con- (Lhermitte-Vallarino and Bain 2004, Lhermitte-Vallarino trary to this, pseudolabia are present in several Rhabdias et al. 2008, 2009a, b), excepting R. ohlerae, which is eas- from bufonids in the Nearctic (Baker 1978), Neotropic ily distinguished by its dilated anterior end. (Kuzmin et al. 2007, Martínez-Salazar and León-Règag- The four Rhabdias species described herein share non 2007) and Oriental region (Lu 1934). a number of biological characters with their congeners A further three species occur in Arthroleptidae (Ta- from other lissamphibian and saurian hosts, such as her- ble 3), with R. collaris and R. ohlerae parasitising Lep- maphrodism in the parasitic females. Male gametes were topelis, and R. tanyai using Astylosternus. Both genera positively identified in the oviducts of R. picardiae and are restricted to sub-Saharan Africa. In Cameroon, R. oh- testis zone in the posterior ovary of R. tanyai, while cells lerae and R. tanyai are morphologically very dissimilar interpreted as such were seen in the oviduct of R. vencesi (Fig. 2). This is not surprising considering that their re- and in the posterior ovary of R. ohlerae. Contrary to Baker spective hosts, Le. brevirostris and As. rheophilus, were (1979), however, who observed protandry in R. america- collected on two isolated mountains of the Cameroonese nus and R. ranae, this was not seen in the present material. volcanic chain, where endemicity is high in plants as well Rather spermatozoa seem to be produced intermittently as animals (Letouzey 1985, Amiet 1987, Vivien 1991, during the females’ entire life, as in some Rhabdias from Maley 1996, Küper et al. 2004). Interestingly, both R. col- other lissamphibian (Travassos 1926, Chu 1936) and cha- laris and R. ohlerae, collected from allopatric forest spe- meleon hosts (Lhermitte-Vallarino and Bain 2004, Lher- cies of the genus Leptopelis, have cephalic dilatations and mitte-Vallarino et al. 2008, 2009a, b). Oviduct structure are close in other aspects of their morphology as well. was uniform in the four new species, consisting of high, Since R. androgyna (Kloss 1971) and R. sphaerocephala narrow cells as in Rhabdias species from Chamaeleoni- (Goodey 1924a) from Bufonidae in Brazil and Europe, dae. No seminal receptacle as described by Baker (1979) respectively, possess similar dilatations without sharing in oviducts of young adults of R. americanus was identi- any further resemblances, it is difficult to judge whether fied. cephalic inflations indicate a common ancestry or are the Additional biological traits shared by R. vencesi, for result of convergent development preventing expulsion which part of the life history could be studied, and Rhab- from the host’s lungs (Baker 1987b). dias from other lissamphibians and saurians, include A single species, R. vencesi, was recovered from Man- heterogonic free-living males and females. Compared to tellidae (Boophis), a family restricted to Madagascar and species from chamaeleonids, the male caudal filament is Mayotte (Frost 2009). In the same region, R. madagas- almost twice as long (Lhermitte-Vallarino and Bain 2004, cariensis was described from Ptychadena. The family Lhermitte-Vallarino et al. 2008, 2009a). Ptychadenidae was historically restricted to sub-Saha- Two morphological characters of the infective stage are ran Africa, but the genus Ptychadena has subsequently of interest: the ornamentation of the sheath and the shape been introduced on Madagascar, the Seychelles and the of the tail extremity of the larva. Rhabdias vencesi shares Mascarene Islands. Both Malagasy Rhabdias species the checkered sheath of its infective larva with congeners are closely related, suggesting speciation through host- from chamaeleonids (Lhermitte-Vallarino et al. 2004, switching, a process that occurred in many groups of 2005, 2008, 2009a), R. fuscovenosa from a snake (Good- nematode parasites (Durette-Desset et al. 1985, Guerrero ey 1924b) and infective larvae of other Rhabdiasidae, et al. 2002). such as Entomelas entomelas (Dujardin, 1845) (Seurat All Rhabdias species from anuran hosts described here- 1920), Pneumonema tiliquae Johnston, 1916 (Ballantyne in differ from the large species of Rhabdias in African and 1991) and Chabirenia cayennensis Lhermitte-Vallarino, Malagasy chameleons in the size of the buccal capsule. Bain, Bertani, Voza, Attout et Gaucher, 2005 (Lhermitte- In R. chamaeleonis (Skrjabin, 1916), R. jarki Lhermitte- Vallarino et al. 2005). The ornamentation of the sheath Vallarino et Bain, 2004, R. cristati Lhermitte-Vallarino et is easily missed. Its description in several genera and Bain, 2008, R. okuensis Lhermitte-Vallarino et Bain, 2008, species of Rhabdiasidae suggests that it is a character of R. brevicorne Lhermitte-Vallarino, Junker et Bain, 2009, Rhabdiasidae. R. mariauxi Lhermitte-Vallarino et Bain, 2009, R. nasutum The tail extremity of the infective larva of R. vencesi, Lhermitte-Vallarino, Junker et Bain, 2009, R. rabetafikae the first lung worm from Mantellidae, is pointed. In four Lhermitte-Vallarino, Junker et Bain, 2009 and R. rhampho- of five species from bufonid and ranid anurans, for which leonis Lhermitte-Vallarino et Bain, 2009 the buccal capsule this character is sufficiently described or illustrated, the is distinctly wider (≥35) than that seen in Rhabdias from tail extremity is blunt (Yuen 1965, Baker 1979), but point- anurans in the Afrotropical region (Table 3). Contrary to ed in the remaining one (Travassos 1926). It is also blunt this, the buccal capsule of the two small species from chain species from ophidians (Kuzmin 1999, Kuzmin and meleons, R. gemellipara Chabaud, Brygoo et Petter, 1961 Miskov 1999). The bifid tail reported by Chu (1936) is the and Rhabdias sp. of Lhermitte-Vallarino et al. (2009), is result of confusion with infective larvae of Strongyloides

59 Grassi, 1879. The four recently studied Rhabdias species Curien, Programme Galileo. We thank Ivo Melle Ngwese, our from chamaeleonids present a particular morphology in field guide, and Laure Pierre who is in charge of the MNHN rep- that the tail is blunt and carries a small number of buds tile collection. We also thank the Cameroon Ministry of Forests (Lhermitte-Vallarino et al. 2004, 2005, 2008, 2009a). and Fauna and Ministry of Scientific Research and Innovation for their ongoing support of research efforts in Cameroon. The Acknowledgements. This work was supported by the Agence visit of K. Junker to the Muséum National d’Histoire Naturelle, Nationale de la Recherche Biodoversity Project, Iles Forestières Paris was financed by the National Research Foundation, South Africaines, IFORA, 2006–2009, and by the Partenariat Hubert Africa (KISC grant No. 67445).

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Received 27 September 2009 Accepted 15 December 2009